Our appreciation of the function of "regulator of G-protein signaling" (RGS) proteins in G protein-coupled signal transduction has shifted since their discovery, from simple G-alpha-subunit GTPase accelerators to higher-order regulators of signal onset, termination, and coordination. The most radical shifts have come from identifying novel functional domains with two RGS protein subfamilies. R7-subfamily proteins each contain a G-gamma-like (GGL) domain which binds G-beta5 subunits and facilitates receptor/G-alpha coupling, whereas R12-subfamily proteins each contain a GoLoco motif which binds G-alpha-i-GDP subunits to inhibit nucleotide release. These novel domains participate in unique G-protein interactions not currently accommodated by the standard model of heterotrimeric G-protein signaling. Thus, to understand the structural bases for these unique interactions, as well as their functional consequences on G-protein signaling, two specific aims are proposed centered around atomic-resolution structural determinations using X-ray crystallography. The first aim is to determine the structural features of the GGL/G-beta5 dimer that support (a) selective association solely with G-beta5, (b) interaction of G-beta5/R7 dimers with GDP-bound G-alpha subunits, and (c) coupling of G-beta5/R7 dimers to receptors. This will be accomplished by solving the crystal structure of a G-beta5/GGL dimer, as well as by extensive mutagenesis of G-beta5/R7 complexes and functional assessments of altered G-alpha-, lipid-, and receptor-binding properties via surface-plasmon resonance biosensor assays and measurements of agonist-dependent receptor/G-protein activation in proteoliposome reconstitutions. The second aim is to determine the structural basis for inhibition of G-alpha-GDP nucleotide dissociation by GoLoco motifs and the structural connections between RGS-boxes and GoLoco regions within R12-family RGS proteins. This will be accomplished by solving crystal structures of (a) a GoLoco motif peptide in complex with a GDP-bound G-alpha-i subunit and (b) G-alpha subunits in complex with larger spans of R12-family proteins that encompass both RGS-box and GoLoco-motif regions. These structural studies will further our understanding of how multi-functional RGS proteins assemble signaling scaffolds to generate highly-specific biological outcomes from seemingly generic and universal intracellular signal transduction modules. Such detailed structural knowledge should lead to identifying novel drug discovery targets of exquisite specificity for the many diseases that involve aberrant G-protein signaling.